The potency of mitochondria enlargement for mitochondria-mediated terpenoid production in yeast

Abstract Terpenoids are widely used in the food, beverage, cosmetics, and pharmaceutical industries. Microorganisms have been extensively studied for terpenoid production. In yeast, the introduction of the mevalonate (MVA) pathway in organelles in addition to the augmentation of its own MVA pathway have been challenging. Introduction of the MVA pathway into mitochondria is considered a promising approach for terpenoid production because acetyl-CoA, the starting molecule of the MVA pathway, is abundant in mitochondria. However, mitochondria comprise only a small percentage of the entire cell. Therefore, we hypothesized that increasing the total mitochondrial volume per cell would increase terpenoid production. First, we ascertained that the amounts of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the final molecules of the MVA pathway, were 15-fold higher of the strain expressing the MVA pathway in mitochondria than in the wild-type yeast strain. Second, we found that different deletion mutants induced different mitochondrial volumes by measuring the mitochondrial volume in various deletion mutants affecting mitochondrial morphology; for example,Δmdm32 increased mitochondrial volume, and Δfzo1 decreased it. Finally, the effects of mitochondrial volume on amounts of IPP/DMAPP and terpenoids (squalene or β-carotene) were investigated using mutants harboring large or small mitochondria expressing the MVA pathway in mitochondria. Amounts of IPP/DMAPP and terpenoids (squalene or β-carotene) increased when the mitochondrial volume expanded. Introducing the MVA pathway into mitochondria for terpenoid production in yeast may become more attractive by enlarging the mitochondrial volume. Key points • IPP/DMAPP content increased in the strain expressing the MVA pathway in mitochondria • IPP/DMAPP and terpenoid contents are positively correlated with mitochondrial volume • Enlarging the mitochondria may improve mitochondria-mediated terpenoid production Supplementary Information The online version contains supplementary material available at 10.1007/s00253-023-12922-5.


Plasmid construction
The plasmids, the primers and the DNA fragments used in this study are listed in Table S1, S2 and S3 respectively.Unless otherwise noted, all of the plasmids were constructed by using In-fusion HD cloning Kit (Takara Bio USA, Mountain View, CA, USA), according to the manufacturer's protocol.

pGK426-MLS-GFP, the plasmid expressing the MLS-GFP
A DNA fragment 1 of partial MLS-fused GFP was amplified using primer 1 and 3 and a template, pGK416-ymUkG1 (Kaishima 2016).Using primer 2 and 3 and the DNA fragment 1 amplified as the template, the DNA fragment 2 of the MLS-GFP was amplified and cloned into pGK426 at the site between SalI and BamHI.

pPreARS208, the plasmid to prepare for construction of pARS208-ERG13-ERG10
The ARS208 upstream region, promoter and terminator set (TDH3 promoter, TDH3 terminator, ADH1 promoter and ADH1 terminator) and ARS208 downstream region were amplified using S.cereviciae BY4741 genome and pATP426 (Ishii 2014) as the template and primer 4 to 9. The DNA fragments 3 to 5 were cloned to pUC19 digested by HindⅢ and NdeI.The resulting plasmid was designated as pPreARS208.

URA3
A DNA fragment 6 of partial MLS-fused ERG13 was amplified using primer 20 and 21 and BY4741 genome as a template.Using primer 20 and 22 and the DNA fragment 7 amplified as the template, the DNA fragment of the MLS-ERG13 was amplified.Similarly, a DNA fragment 8 of partial MLS-fused ERG10 was amplified using primer 25 and 27 and BY4741 genome, and, using primer 26 and 27 and the DNA fragment 8 amplified as the template, the DNA fragment 9 of the MLS-ERG10 was amplified.
The DNA fragment 10 containing TDH3 promoter for the MLS-ERG13 and ADH1 promoter for the MLS-ERG10 was amplified using primer 23 and 24 and pATP426 as the template.The DNA fragments 7,9 and 10 were cloned to pPre208 digested by PmeI and MluI.The resulting plasmid was designated as pARS208-ERG13-ERG10.

pARS208-ERG13-ERG10-URA3, the plasmid for integration of the genes encoding MLS-ERG13 and MLS-ERG10 into genome
The DNA fragment 11 of the URA3 gene was amplified using primer 36 and 37 and pGK426 as the template.

pPreARS308, the plasmid to prepare for construction of pARS308-ERG12-tHMG1
The ARS308 upstream region, promoter and terminator set (TDH3 promoter, TDH3 terminator, ADH1 promoter and ADH1 terminator) and ARS308 downstream region were amplified using S.cereviciae BY4741 genome and pATP426 as the template and primer 7 and 11 to 14.The DNA fragments 12 to 14 were cloned to pUC19 digested by HindⅢ and NdeI.The resulting plasmid was designated as pPreARS308.

URA3
A DNA fragment 15 of partial MLS-fused ERG12 was amplified using primer 28 and 29 and BY4741 genome as a template.Using primer 22 and 28 and the DNA fragment 15 amplified as the template, the DNA fragment 16 of the MLS-ERG12 was amplified.Similarly, a DNA fragment 17 of partial MLS-fused tHMG1 was amplified using primer 30 and 31 and BY4741 genome, and, using primer 26 and 31 and the DNA fragment 17 amplified as the template, the DNA fragment 18 of the MLS-tHMG1 was amplified.
The DNA fragment 10 containing TDH3 promoter for the MLS-ERG12 and ADH1 promoter for the MLS-tHMG1 was amplified using primer 23 and 24 and pATP426 as the template.The DNA fragments 10,16 and 18 were cloned to pPre308 digested by PmeI and MluI.The resulting plasmid was designated as pARS308-ERG12-tHMG1.

ERG12 and MLS-tHMG1 into genome
The DNA fragment 11 of the URA3 gene was amplified using primer 36 and 37 and pGK426 as the template.

pPreARS416, the plasmid to prepare for construction of pARS416-ERG19-ERG8
The ARS416 upstream region, promoter and terminator set (TDH3 promoter, TDH3 terminator, ADH1 promoter and ADH1 terminator) and ARS416 downstream region were amplified using S.cereviciae BY4741 genome and pATP426 as the template and primer 7 and 15 to 19.The DNA fragments 19 to 21 were cloned to pUC19 digested by HindⅢ and NdeI.The resulting plasmid was designated as pPreARS416.

URA3
A DNA fragment 22 of partial MLS-fused ERG19 was amplified using primer 32 and 33 and BY4741 genome as a template.Using primer 22 and 32 and the DNA fragment 22 amplified as the template, the DNA fragment 23 of the MLS-ERG19 was amplified.Similarly, a DNA fragment 24 of partial MLS-fused ERG8 was amplified using primer 34 and 35 and BY4741 genome, and, using primer 26 and 35 and the DNA fragment 24 amplified as the template, the DNA fragment 25 of the MLS-ERG8 was amplified.
The DNA fragment 26 containing TDH3 promoter for the MLS-ERG19 and ADH1 promoter for the MLS-ERG8 was amplified using primer 23 and 24 and pATP426 as the template.The DNA fragments 10, 23 and were cloned to pPre308 digested by PmeI and MluI.The resulting plasmid was designated as pARS416-ERG19-ERG8.

and MLS-ERG8 into genome
The DNA fragment 11 of the URA3 gene was amplified using primer 36 and 37 and pGK426 as the template.

pFzo1, the plasmid to prepare for construction of pFzo1-URA3 and remove URA3 integrated into genome
The Fzo1 upstream region and downstream region were amplified using S.cereviciae BY4741 genome and primer 40 to 43.The DNA fragments 26 and 27 were cloned to pUC19 digested by digested by HindⅢ and NdeI.The resulting plasmid was designated as pFzo1.

pFzo1-URA3, the plasmid for deletion of Fzo1
The Fzo1 upstream region, URA3 gene and Fzo1downstream region were amplified using S.cereviciae BY4741 genome and primer 40, 43 56 to 59.The DNA fragments 28 to 30 were cloned to pUC19 digested by digested by HindⅢ and NdeI.The resulting plasmid was designated as pFzo1-URA3.

pMgm1, the plasmid to prepare for construction of pMgm1-URA3 and remove URA3 integrated into genome
The Mgm1 upstream region and downstream region were amplified using S.cereviciae BY4741 genome and primer 44 to 47.The DNA fragments 31 and 32 were cloned to pUC19 digested by digested by HindⅢ and NdeI.The resulting plasmid was designated as pMgm1.

pUgo1, the plasmid to prepare for construction of pUgo1-URA3 and remove URA3 integrated into genome
The Ugo1 upstream region and Ugo1 downstream region were amplified using S.cereviciae BY4741 genome and primer 48 to 51.The DNA fragments 35 and 36 were cloned to pUC19 digested by digested by HindⅢ and NdeI.The resulting plasmid was designated as pUgo1.

pMdm32, the plasmid to prepare for construction of pMdm32-URA3 and remove URA3 integrated into genome
The Mdm32 upstream region and downstream region were amplified using S.cereviciae BY4741 genome and primer 52 to 55.The DNA fragmnts 39 and 40 were cloned to pUC19 digested by digested by HindⅢ and NdeI.The resulting plasmid was designated as pMdm321.

pCrtYBI-BTS1, the plasmid for expressing CrtYBI and overexpressing BTS1
The hygromycin B resistance gene region was amplified using pRDH227 plasmid and primer 74 and 75.
The DNA fragment was cloned to pATP416-crtYBI digested by NsiI and SbfI.The resulting plasmid was designated as pCrtYBI-BTS1.

Construction of recombinant yeast strain
Yeast strains used in this study are listed in Table 1.S.cereviciae transformation was carried out by using a lithium acetate transformation method, as previously reported (Chen 1992) .The resulting transformants were spread on SD-URA or SD with 5-fluoroorotic acid (5-FOA) agar plate.

Construction of SSY2 to SSY5
A DNA fragment 44 composed of the Fzo1 upstream region, URA3 gene and Fzo1 downstream region was amplified using pFzo1-URA3 as the template and primer 66 and 67, transfected into the SSY1, yielding SSY1 DFzo1::URA3.Then, a DNA fragment 45 of Fzo1 upstream region and downstream region was amplified using pFzo1 as the template and primer 66 and 67.To remove the URA3 gene from SSY1 DFzo1::URA3, the DNA fragment 45 was transfected into the SSY1 DFzo1::URA3 and SSY2 was obtained.
The same method was applied using the DNA fragments 46 and 47 or 48 and 49 or 50 and 51 to construct SSY3 to SSY5.

Fig. S5
Fig. S5The growth of SSY2 was not improved by DTT addition

Table S3
The DNA fragments list 187